Porcine circovirus type 2 vlp vaccine

ABSTRACT

Provided is PCV2 VLP that can be used as a vaccine for use in the field of animal husbandry and that has high molecular stability against physicochemical load. A fusion protein according to the present invention includes a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain.

TECHNICAL FIELD

The present invention relates to a fusion protein, virus-like particles (VLP) containing the fusion protein, and a vaccine containing the VLP. The present invention also relates to a method for producing the fusion protein.

BACKGROUND ART

Porcine circovirus type 2 (porcine circovirus 2 (PCV2)) induces immunodeficiency of swine infected therewith and increases susceptibility to other infectious diseases. Thus, anti-PCV2 measures are important for pig farmers. Swine infected with PCV2 develop porcine circovirus associated diseases (PCVAD). A major symptom of PCVAD is postweaning multisystemic wasting syndrome (PMWS). PMWS occurs more often in 5 to 6-week old swine and is clinically characterized by wasting, paleness of the skin, stunted growth, respiratory distress, diarrhea, and jaundice (Non-patent Literature 2). PCV2, as the causative virus of PCVAD, was first isolated from swine that developed PMWS in western Canada (Non-patent Literature 3).

PCV2 is a non-enveloped virus with a diameter of approximately 20 nm which contains single-stranded circular DNA in its genome. PCV2 has three open reading frames (ORFs: ORF1, ORF2, and ORF3) in which ORF2 encodes for a capsid protein (Cap). It is known that 60 monomers of Cap associate and form a virus-like particle as a regular icosahedral multimeric molecule (Non-patent Literature 1). Since the VLP does not contain viral genomic DNA and thus cannot proliferate, the VLP can be applied to a vaccine for preventing PCV2 infection.

As the vaccine for preventing PCV2 infection, the PCV2 inactivated vaccine “Circovac (registered trademark)” and the PCV1/PCV2 chimeric virus inactivated vaccine “Suvaxyn (registered trademark) PCV2” have been launched on the market. In addition to these inactivated vaccines, for example, the VLP vaccines “Posilis (registered trademark) PCV” (Non-patent Literature 4) and “Ingelvac CircoFLEX (registered trademark)” (Patent Literature 1), each of which contains Cap produced in a recombinant baculovirus/insect cell expression system, have been put into practical use.

Furthermore, attempts have been made to produce Cap of PCV2 using, for example, a Pichia yeast (Pichia pastoris), a budding yeast (Saccharomyces cerevisiae), and Escherichia coli as an expression system (Non-patent Literatures 1 and 5 to 13).

Non-patent Literature 16 has reported that antigens fused to immunoglobulin-binding domains (IBDs) were efficiently transported to lymphoid follicles and had improved immunogenicity.

Further, Non-patent Literature 17 describes a Z domain as an IBD. The Z domain is an IBD derived from Staphylococcus aureus protein A and has a protein three-dimensional structure, called a three-helix bundle, in which three α-helices are folded and bundled. Note that protein A is one of the virulence factors of Staphylococcus aureus and is a 42 kDa protein present on an outer membrane of a bacterial cell. Also, protein A has five IBDs (E, D, A, B and C domains) having high similarity in primary sequence. Note that the Z domain is an artificial sequence prepared by introducing two amino acid substitutions into the B domain.

CITATION LIST Patent Literatures [Patent Literature 1]

-   Japanese Translation of PCT International Application, Tokuhyo, No.     2008-526212

Non-Patent Literatures [Non-patent Literature 1]

-   Khayat R. et al., J. Virol. 2011, 85, 7856-7862

[Non-patent Literature 2]

-   Harding J.C.S. et al., Swine Health Prod. 1997, 5, 201-203

[Non-patent Literature 3]

-   Ellis J. et al., Can. Vet. J. 1998, 39, 44-51

[Non-patent Literature 4]

-   Liu L.-J. et al., Arch. Virol. 2008, 153, 2291-2295

[Non-patent Literature 5]

-   Tu Y. et al., Appl. Microbiol. Biotechnol. 2013, 97, 2867-2875

[Non-patent Literature 6]

-   Zaveckas M. et al., J. Chromatogr. B 2015, 991, 21-28

[Non-patent Literature 7]

-   Marcekova Z. et al., J. Virol. Methods 2009, 162, 133-141

[Non-patent Literature 8]

-   Liu Q. et al., Protein Expr. Purif. 2001, 21, 115-120

[Non-patent Literature 9]

-   Yin S. et al., Virol. J. 2010, 7, 166

[Non-patent Literature 10]

-   Wu P C et al., Appl. Microbiol. Biotechnol. 2012, 95, 1501-1507

[Non-patent Literature 11]

-   Wu P C et al., J. Biotechnol. 2016, 220, 78-85

[Non-patent Literature 12]

-   Zhang Y. et al., Arch. Virol. 2016, 161, 1485-1491

[Non-patent Literature 13]

-   Xi X. et al., J. Biotechnol. 2016, 223, 8-12

[Non-patent Literature 14]

-   Wang N. et al., J. Virol. Methods 2017, 243, 146-150

[Non-patent Literature 15]

-   Burroughs A. M. et al., Biol. Direct 2007, 2, 18

[Non-patent Literature 16]

-   Miyata T. et al., Infect. Immun. 2011, 79, 4260-4275

[Non-patent Literature 17]

-   Bjorn N. et al., Protein Eng. 1987, 1, 107-113

SUMMARY OF INVENTION Technical Problem

As mentioned above, some vaccines have already been launched on the market as agents for prevention of PCV2 infection, but PCV2 has a problem that PCV2-infected cultured cells are difficult to obtain a high virus titer.

Further, using the recombinant baculovirus/insect cell expression system as the expression system for producing Cap of PCV2 has problems in terms of cost and work complexity. In addition, VLP formed from Cap produced in such an expression system has a problem in terms of molecular stability against physicochemical load, and there is a problem that it is difficult to stably store the VLP for a long period of time.

Furthermore, in a case where an Escherichia coli expression system is used as the expression system for producing Cap of PCV2, expressed proteins become insoluble aggregates (inclusion bodies) and accumulate in the bacterial cells. Therefore, there have been no reports of VLP being spontaneously reconstituted (spontaneously associated) in the cytoplasm of Escherichia coli and being collected as a soluble VLP protein.

Therefore, there has been a demand for further development of PCV2 vaccines.

The present invention has been attained in view of the above-described problems, and an object of the present invention is to provide (i) PCV2 VLP that can be used as a vaccine for use in the field of animal husbandry and that has high molecular stability against physicochemical load and (ii) a technique using the PCV2 VLP.

Solution to Problem

As a result of diligent study in view of the above-described problems, the inventors of the present invention have completed the present invention after newly finding, for example, that (i) a fusion protein of PCV2 Cap and IBD (Cap-IBD fusion protein) is capable of spontaneously associating in the cytoplasm of an Escherichia coli expression host and forming VLP without undergoing an artificial association process in vitro, (ii) the VLP thus formed has molecular stability against physicochemical load such as heat treatment and long-term low-temperature/freezing storage, and (iii) the obtained VLP can be used as a vaccine.

That is, an aspect of the present invention relates to a fusion protein including a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain.

Further, an aspect of the present invention relates to a method for producing a fusion protein including a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain, the method including the steps of: preparing an expression vector containing a nucleic acid which encodes the fusion protein including the capsid protein of the porcine circovirus type 2 and the immunoglobulin binding domain; and introducing the expression vector into Escherichia coli to express the fusion protein.

Advantageous Effects of Invention

According to an aspect of the present invention, it is possible to provide (i) PCV2 VLP that can be used as a vaccine for use in the field of animal husbandry and that has high molecular stability against physicochemical load and (ii) a technique using the PCV2 VLP.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a conceptual diagram for explaining the problems of the conventional techniques and the features of the present invention on VLP formation from PCV2 Cap.

FIG. 2 is a schematic view of a protein structure, a β-grasp fold structure, and a three-helix bundle structure of a solubilization-promoting tag (SUMO) and IBDs (SpGB, PpLB, Z).

FIG. 3 is a schematic view illustrating the arrangements of Cap and IBDs (SpGB1-B2, PpLB1-B2, ZZ) or the solubilization-promoting tag (SUMO) in the fusion proteins collected in Examples.

FIG. 4 is an electron micrograph of VLP of PCV2 Cap-SpGB1-B2.

FIG. 5 is an electron micrograph of VLP of PCV2 Cap-PpLB1-B2.

FIG. 6 is an electron micrograph of VLP of PCV2 Cap-ZZ.

FIG. 7 is an electron micrograph of VLP of PCV2 Cap-SUMO.

FIG. 8 is a chromatogram representing the analysis results obtained by gel filtration chromatography before and after heat treatment, for VLP of PCV2 Cap-ZZ.

FIG. 9 is a chromatogram representing the analysis results obtained by gel filtration chromatography before and after heat treatment, for VLP of PCV2 Cap-ZZ after subjected to dissociation of ZZ from the VLP surface layer of PCV2 Cap-ZZ.

FIG. 10 is an electron micrograph of VLP of PCV2 Cap-ZZ after stored in a PBS solution at 4° C. for 1 year.

FIG. 11 is an electron micrograph of VLP of PCV2 Cap-ZZ after stored in a PBS solution at −30° C. for 1 year.

FIG. 12 is an electron micrograph of VLP of PCV2 Cap-ZZ after stored in a PBS solution at −80° C. for 1 year.

FIG. 13 is a graph showing the results of measurement of a viral copy number in serum.

FIG. 14 is a graph showing the results of measurement of a viral copy number in lymph nodes.

DESCRIPTION OF EMBODIMENTS Problems of the Conventional Techniques and Outline of the Present Invention

As mentioned above, the conventional techniques regarding the PCV2 vaccine have various problems.

For example, as reported in Non-patent Literature 14, the problem with the use of an Escherichia coli expression system is that the VLPs formed from the Caps which have been produced in the Escherichia coli expression system are molecules which are unstable against physicochemical load. According to Non-patent Literature 14, when the VLPs from the PCV2 Caps are stored at 4° C. in a PBS solution, their particle shape cannot be maintained even for only one month, and for long-term storage, it is necessary to use a special solution in which a plurality of compounds including 15% trehalose and the like are mixed.

Further, in the Pichia yeast expression system described in Non-patent Literature 5, there is no evidence that VLPs were formed from the produced Caps. Further, in the budding yeast expression system described in Non-patent Literature 6, the VLPs formed from the produced Caps have distorted shapes.

Furthermore, the Escherichia coli expression systems described in Non-patent Literatures 1 and 7 to 13 are attractive in terms of cost and simplicity of work, but still have many issues to be faced to express structurally complex multimeric molecules like VLPs.

Specifically, most of heterologous proteins expressed in the Escherichia coli expression system often become insoluble aggregates (inclusion bodies) and accumulate in the bacterial cells. Since the heterologous proteins that have become inclusion bodies do not have a natural three-dimensional structure, most of them do not function as effective vaccines. A refolding method is known as a method for rewinding heterologous proteins that have become inclusion bodies into a natural three-dimensional structure. Also in some of the above-described Escherichia coli expression systems, solubilizing the PCV2 Cap having been collected from the inclusion bodies with a denaturing agent, such as guanidine hydrochloride, and refolding the denatured PCV2 Cap by a dialysis method or the like are attempted.

Meanwhile, as a method for expressing a heterologous protein as a soluble protein, a method of fusing a solubilization-promoting tag to a target protein is commonly used. As such a solubilization-promoting tag, for example, glutathione S-transferase (GST), maltose-binding protein (MBP), and small ubiquitin-like modifier (SUMO) having β-grasp fold structure (protein structure like a structure in which one α-helix is grasped by one β-sheet) (Non-patent Literature 15) are known. When these solubilization-promoting tags are fused mainly to an N-terminal of the target protein and expressed, the fusion protein can be collected from a soluble fraction of an Escherichia coli expression host. Also in some of the above-described Escherichia coli expression systems, fusing the solubilization-promoting tag to the N-terminal of the PCV2 Cap to collect the PCV2 Cap as a soluble protein is attempted. Unfortunately, there are many problems in forming VLPs that can function as vaccines from the collected soluble Caps.

For example, in the Escherichia coli expression system described in Non-patent Literature 1, complicated work processes, such as nickel affinity purification and subsequent in vitro buffer exchange, are required to form VLPs from the PCV2 Cap expressed as a soluble protein.

Further, in the Escherichia coli expression system described in Non-patent Literature 7, the PCV2 Cap is also expressed as a soluble protein, but even though the obtained Cap is subjected to post-treatment such as ion exchange chromatography, VLPs have not been formed.

In the Escherichia coli expression system described in Non-patent Literature 8, GST or MBP as the solubilization-promoting tag is fused to the N-terminal of the PCV2 Cap. However, the GST-Cap fusion protein has only been detected as a degradation product. Further, the MBP-Cap fusion protein is expressed as a soluble protein, but do not form VLPs in vitro even after undergoing a subsequent affinity purification step.

In the Escherichia coli expression system described in Non-patent Literature 9, SUMO as a solubilization-promoting tag is fused to an N-terminal of PCV2 Cap. As a result, a soluble SUMO-Cap fusion protein was expressed, but this fusion protein did not form VLPs by spontaneous association (self-association). Thus, formation of VLPs in vitro is attempted by cleaving and removing the SUMO tag with SUMO protease and subjecting the obtained Cap to a complicated post-treatment such as dialysis treatment. However, the formed VLPs are morphologically different from natural PCV2 particles, and have problems such as distorted particle shape and non-uniformity in particle diameter.

In the Escherichia coli expression systems described in Non-patent Literatures 10 and 11, formation of VLPs is attempted by using only a natural Cap sequence that does not contain any fusion tag or the like. However, the amount of VLPs obtained is extremely small, and purification treatment by, for example, a sucrose density gradient centrifugation method (Non-patent Literature 10) or a gel filtration chromatography method (Non-patent Literature 11) is required. Further, the formed VLPs have a morphological problem like the VLPs of Non-patent Literature 9.

In the Escherichia coli expression system described in Non-patent Literature 12, formation of VLPs from an expressed soluble Cap is attempted. However, as in the above case, various complicated work processes are required.

The Escherichia coli expression system described in Non-patent Literature 13 is a method limited to a specific genotype PCV2, and work processes such as buffer exchange is indispensable for promoting formation of VLPs in vitro from the expressed soluble Cap.

That is, the above-described Escherichia coli expression systems of Non-patent Literatures 1 and 7 to 13 attempt to reconstruct VLPs in vitro by fusion of the solubilization-promoting tags or other operations. However, in all of these expression systems, various complicated work processes such as buffer exchange are indispensable. In addition, most of the formed VLPs have problems such as distorted particle shape, non-uniformity in particle diameter, and formation of agglomerates.

In order to produce a vaccine for use in the field of animal husbandry, there has been a demand for development of an Escherichia coli expression system capable of producing VLPs having good morphology which is free from, for example, distorted particle shape, without the need for complicated work processes. One conceivable means for eliminating complicated work processes is to cause a soluble Cap to make spontaneous association (self-association) in the cell of the Escherichia coli expression host so that the Escherichia coli expression host spontaneously forms VLPs. This makes it possible to collect VLPs with good morphology without undergoing an artificial association process in vitro.

It has been reported that natural PCV2 VLP is such that the N-terminal of Cap is located on the luminal side of the particle, and the C-terminal thereof is located on the surface layer side thereof (Non-patent Literature 1). However, since the previously reported solubilization-promoting tag is fused to the N-terminal of Cap, there is a physical limitation for spontaneous association of Cap. Therefore, it is difficult to cause Cap to spontaneously associate in an Escherichia coli expression system using such a solubilization-promoting tag.

The inventors of the present invention, as a result of diligent studies to solve the above-described problems, have obtained new findings and succeeded in developing a novel PCV2 vaccine based on the new findings.

The outline of the present invention will be described with reference to FIG. 1, in comparison with the conventional techniques.

The upper and middle parts of FIG. 1 illustrate the conventional techniques on VLP formation from PCV2 Cap, and the lower part of FIG. 1 illustrates the features of the present invention on VLP formation from PCV2 Cap.

As illustrated in the upper part of FIG. 1, in a PCV2 Cap expression system using an insect cell or a mammalian cell as a host, soluble Caps are expressed, and spontaneously associate (self-associate) in the cell of the expression host to form a VLP. However, application of physicochemical load such as low-temperature long-term storage to the VLP makes the VLP unable to maintain its original particle shape and causes disintegration into Cap monomers and formation of aggregates (Cap aggregates).

Further, as illustrated in the middle part of FIG. 1, in a PCV2 Cap expression system using Escherichia coli as a host, even though Cap is expressed as a soluble protein, spontaneous association (self-association) itself in a cell of the host is very difficult. VLP cannot be formed without complicated work processes in vitro.

In contrast, as illustrated in the lower part of FIG. 1, in the present invention, when a Cap-IBD fusion protein was expressed in a PCV2 Cap expression system using Escherichia coli as a host, it was revealed that Caps spontaneously associate (self-associate) to form VLP in the cell of the expression host, as in the case of the insect cell/mammalian cell expression system. Furthermore, it was also revealed that the formed VLP had good molecular stability (for example, long-term low-temperature storage stability) against physicochemical load, unlike the VLP formed in the above-described insect cell/mammalian cell expression system. However, it was found that when the IBDs were dissociated from a surface layer of the VLP thus formed, the VLP was disintegrated by application of physicochemical load, like the VLP formed in the above-described insect cell/mammalian cell expression system. That is, it was revealed that fusing IBDs to Cap newly imparts (1) “VLP formation promoting function” and (2) “VLP disintegration suppressing function”.

[Fusion Protein and Virus-Like Particle (VLP)]

A fusion protein in accordance with an embodiment of the present invention is a fusion protein including PCV2 Cap and IBD (also referred to as “Cap-IBD fusion protein”).

Further, PCV2 VLP in accordance with an embodiment of the present invention is a multimeric molecule formed by association of a plurality of Cap-IBD fusion proteins.

The Cap-IBD fusion proteins can be expressed as soluble proteins in the Escherichia coli expression system and can spontaneously associate (self-associate) in a cell of the Escherichia coli expression host to spontaneously form a VLP.

As used herein, the term “VLP” refers to a particle that has a shape similar to that of a natural virus and that is a non-infectious particle. The VLP is a particle having a shape similar to that of a natural virus, as described above, and thus exhibits immunological cross-reactivity with the natural virus and functions as an effective vaccine. Since the VLP lacks a viral genome, the VLP is non-proliferative and non-infectious and is extremely safe.

As used herein, the term “Cap” means a capsid protein of PCV2.

In an embodiment of the present invention, Cap can be any of proteins indicated in (1) to (3) below or a protein that is encoded by a gene consisting of a polynucleotide indicated in (4) or (5) below:

(1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;

(2) a protein (i) consisting of an amino acid sequence obtained by deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 and (ii) having the function of forming a VLP;

(3) a protein (i) consisting of an amino acid sequence having a sequence identity of not less than 90% with respect to the amino acid sequence represented by SEQ ID NO: 1 and (ii) having the function of forming a VLP;

(4) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2; or

(5) a polynucleotide (i) being hybridizable, under a stringent condition, with a DNA consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2 and (ii) encoding a protein that has the function of forming a VLP.

With regard to whether or not PCV2 Cap is a protein having the function of forming a VLP, it is possible to determine that PCV2 Cap has the function of forming a VLP, if a VLP is formed when a fusion protein of PCV2 Cap and IBD in an embodiment of the present invention is formed and expressed in Escherichia coli.

The protein (3) above preferably has a higher sequence identity. The sequence identity can be, for example, not less than 91%, not less than 92%, not less than 93%, not less than 94%, not less than 95%, not less than 96%, not less than 97%, not less than 98% or not less than 99%. Note that the sequence identity of amino acid sequences can be calculated by using GENETYX Ver. 14 (product name, manufactured by Genetyx Corporation) according to a manual of the product. Note that the same applies to the identities of proteins (8), (13), and (18), which will be described later.

With regard to the term “amino acid sequence obtained by deletion, substitution, or addition of one or several amino acids” as used herein, a position where one or several amino acids are deleted, substituted or added is not particularly limited.

Further, the number of amino acids intended by the term “one or several amino acids” is not particularly limited, and can be not more than 50, not more than 40, not more than 30, not more than 20, not more than 19, not more than 18, not more than 17, not more than 16, not more than 15, not more than 14, not more than 13, not more than 12, not more than 11, not more than 10, not more than 9, not more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, not more than 2, or 1.

It is preferable that the substitution of an amino acid be a conservative substitution. Note that the term “conservative substitution” refers to a substitution of a particular amino acid by another amino acid having a chemical property and/or a structure that is/are similar to that/those of the particular amino acid. Examples of the chemical property include a degree of hydrophobicity (hydrophobicity and hydrophilicity) and electric charge (neutrality, acidity, and basicity). Examples of the structure include an aromatic ring, an aliphatic hydrocarbon group, and a carboxyl group, which are present as a side chain or as a functional group of a side chain.

Examples of the conservative substitution include a substitution between serine and threonine, a substitution between lysine and arginine, and a substitution between phenylalanine and triptophan. As a matter of course, the substitution in an embodiment of the present invention is not limited to the above-described substitutions.

The term “stringent condition” as used herein refers to a condition under which (i) a double-stranded polynucleotide specific to a nucleotide sequence is formed, and (ii) a non-specific double-stranded polynucleotide is not formed. In other words, the stringent condition can be expressed as a condition under which hybridization is carried out at a temperature in a range from (i) a melting temperature (Tm) of nucleic acids having a high homology (e.g., a perfectly-matched hybrid) to (ii) 15° C. lower than the melting temperature (Tm), preferably 10° C. lower than the melting temperature (Tm), and further preferably 5° C. lower than the melting temperature (Tm).

In one example of the stringent condition, hybridization can be carried out in a buffer solution (consisting of 0.25M Na₂HPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1×Denhardt's solution) for 16 hours to 24 hours at a temperature in a range from 60° C. to 68° C., preferably at 65° C., and further preferably at 68° C., and then washing can be carried out twice in a buffer solution (consisting of 20 mM Na₂HPO₄, pH 7.2, 1% SDS, and 1 mM EDTA) for 15 minutes at a temperature in a range from 60° C. to 68° C., preferably at 65° C., and further preferably at 68° C.

In another example, prehybridization is carried out overnight at 42° C. in a hybridization solution (including 25% formamide or 50% formamide (for a severer condition), 4×SSC (sodium chloride/sodium citrate), 50 mM Hepes pH 7.0, 10×Denhardt's solution, and 20 μg/ml denatured salmon sperm DNA), and then hybridization is carried out by adding a labeled probe thereto and keeping a resulting solution at 42° C. overnight. In washing following the hybridization, conditions for a washing solution and a temperature are approximately “1×SSC, 0.1% SDS, 37° C.”, approximately “0.5×SSC, 0.1% SDS, 42° C.” for a severer condition, approximately “0.2×SSC, 0.1% SDS, 65° C.” for a further severer condition. As such, as the conditions for the washing following the hybridization become severer, the specificity of hybridization becomes higher. However, the above-indicated combinations of conditions on SSC, SDS, and temperature are merely examples. A person skilled in the art can provide a stringency similar to the above by appropriately combining the above-described or other elements (e.g., a probe concentration, a probe length, and a time period for a hybridization reaction) that determine the stringency of hybridization. This is disclosed in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

As used herein, the term “IBD” refers to a functional unit of a polypeptide having an immunoglobulin-binding ability. The IBD suitably used in the present invention includes IBD having a β-grasp fold structure and IBD having a three-helix bundle structure.

Examples of the IBD having a β-grasp fold structure include a B domain of protein G (SpGB) and a B domain of protein L (PpLB).

Examples of SpGB include a B1 domain and a B2 domain of protein G. In an embodiment of the present invention, a domain in which a B1 domain and a B2 domain are linked can be particularly preferably used as SpGB.

In an embodiment of the present invention, SpGB can be any of proteins indicated in (6) to (8) below or a protein that is encoded by a gene consisting of a polynucleotide indicated in (9) or (10) below:

(6) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 5;

(7) a protein (i) consisting of an amino acid sequence obtained by deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 3 or 5 and (ii) having the function of forming a VLP;

(8) a protein (i) consisting of an amino acid sequence having a sequence identity of not less than 90% with respect to the amino acid sequence represented by SEQ ID NO: 3 or 5 and (ii) having the function of forming a VLP;

(9) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4 or 6; or

(10) a polynucleotide (i) being hybridizable, under a stringent condition, with a DNA consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 4 or 6 and (ii) encoding a protein that has the function of forming a VLP.

With regard to whether or not SpGB is a protein having the function of forming a VLP, it is possible to determine that SpGB has the function of forming a VLP, if a VLP is formed when a fusion protein of SpGB and PCV2 Cap in an embodiment of the present invention is formed and expressed in Escherichia coli.

Examples of PpLB include a B1 domain and a B2 domain of protein L. In an embodiment of the present invention, a domain in which a B1 domain and a B2 domain are linked can be particularly preferably used as PpLB.

In an embodiment of the present invention, PpLB can be any of proteins indicated in (11) to (13) below or a protein that is encoded by a gene consisting of a polynucleotide indicated in (14) or (15) below:

(11) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7 or 9;

(12) a protein (i) consisting of an amino acid sequence obtained by deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 7 or 9 and (ii) having the function of forming a VLP;

(13) a protein (i) consisting of an amino acid sequence having a sequence identity of not less than 90% with respect to the amino acid sequence represented by SEQ ID NO: 7 or 9 and (ii) having the function of forming a VLP;

(14) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8 or 10; or

(15) a polynucleotide (i) being hybridizable, under a stringent condition, with a DNA consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 8 or 10 and (ii) encoding a protein that has the function of forming a VLP.

With regard to whether or not PpLB is a protein having the function of forming a VLP, it is possible to determine that PpLB has the function of forming a VLP, if a VLP is formed when a fusion protein of PpLB and PCV2 Cap in an embodiment of the present invention is formed and expressed in Escherichia coli.

Examples of the IBD having a three-helix bundle structure include a Z domain (Z) of protein A. Z is an artificial polypeptide, described in Non-patent Literature 17 and others, produced by introducing two amino acid substitutions into the B domain of protein A.

In an embodiment of the present invention, Z can be any of proteins indicated in (16) to (18) below or a protein that is encoded by a gene consisting of a polynucleotide indicated in (19) or (20) below:

(16) a protein consisting of the amino acid sequence represented by SEQ ID NO: 11;

(17) a protein (i) consisting of an amino acid sequence obtained by deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 11 and (ii) having the function of forming a VLP;

(18) a protein (i) consisting of an amino acid sequence having a sequence identity of not less than 90% with respect to the amino acid sequence represented by SEQ ID NO: 11 and (ii) having the function of forming a VLP;

(19) a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12; or

(20) a polynucleotide (i) being hybridizable, under a stringent condition, with a DNA consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12 and (ii) encoding a protein that has the function of forming a VLP.

With regard to whether or not Z is a protein having the function of forming a VLP, it is possible to determine that Z has the function of forming a VLP, if a VLP is formed when a fusion protein of Z and PCV2 Cap in an embodiment of the present invention is formed and expressed in Escherichia coli.

In an embodiment of the present invention, an IBD fused to Cap preferably contains a domain selected from the group consisting of SpGB, PpLB, and Z, and particularly preferably contains Z.

FIG. 2 illustrates a schematic view of a protein structure, a β-grasp fold structure, and a three-helix bundle structure of a solubilization-promoting tag (SUMO) and IBDs (SpGB, PpLB, Z). As illustrated in FIG. 2, SUMO (96 amino acid residues), which is a known solubilization-promoting tag, has a β-grasp fold structure, as in the case of SpGB and PpLB. SpGB, PpLB, and Z are each composed of 55 to 60 amino acid residues, and have a primary structure of about half the size of a primary structure of SUMO.

It has been revealed by the present invention that the above-described IBDs have a function of maturing Cap when fused with Cap, which is completely different function from the original antibody-binding ability. The term “maturation” of Cap as used herein means that Cap makes spontaneous association (self-association) in the cytoplasm of an Escherichia coli expression host to form a VLP that morphologically strongly resembles natural PCV2 particles. Such a VLP can be suitably used as a vaccine for preventing PCV2 infection. Further, by administering or inoculating the target animal with a vaccine containing the VLP, it is possible to induce immunity having high antigen specificity against infectious PCV2 and acquire the ability to induce a virus neutralizing antibody. As a result, vaccination of PCV2 VLP makes it possible to effectively suppress PCV2 infection of the target animal.

In a preferable embodiment of the present invention, the IBD constituting the Cap-IBD fusion protein has a structure in which two domains of the same type are linked. Examples of such a structure include, for example, a structure in which two SpGBs are linked (SpGB1-B2: SEQ ID NO: 13 (amino acid sequence), SEQ ID NO: 14 (nucleotide sequence)), a structure in which two PpLBs are linked (PpLB1-B2: SEQ ID NO: 15 (amino acid sequence), SEQ ID NO: 16 (nucleotide sequence)), a structure in which two Zs are linked (ZZ: SEQ ID NO: 17 (amino acid sequence), SEQ ID NO: 18 (nucleotide sequence)), and the like structure. By using SpGB1-B2, PpLB1-B2, or ZZ as the IBD constituting the Cap-IBD fusion protein, it is possible to further enhance the stability of the fusion protein as a soluble protein. This is presumed to be because that the IBD formed by linking two SpGBs, PpLBs or Zs has a molecular weight equivalent to that of the solubility-promoting tag (SUMO) and thus has acquired a solubilization promoting function comparable to that of SUMO. In a case where a plurality of domains of the same type are linked in the IBD, the number of the linked domains is not limited to two, and may be, for example, three, four, five, or more.

In an embodiment of the present invention, the IBD in the Cap-IBD fusion protein is preferably bound to a C-terminal of Cap. With the IBD bound to the C-terminal of Cap, the VLP formation promoting function and VLP disintegration suppressing function of the IBD are further improved. That is, in the cell of the Escherichia coli expression host, the Cap-IBD fusion protein spontaneously (self) associates and forms VLP such that the N-terminal is located on the luminal side of the particle and the C-terminal is located on the surface layer side of the particle. The formed VLP has IBDs on the surface layer thereof and has good molecular stability against physicochemical load.

In an embodiment of the present invention, the binding between Cap and the IBD, and the linkage between the domains may be made via a linker consisting of an appropriate amino acid sequence.

The linker used in an embodiment of the present invention is not particularly limited, but preferably includes any peptide consisting of 4 to 10 or more amino acid residues. Examples of such a peptide include commonly used peptides such as GPGP (4 amino acid residues, SEQ ID NO: 19), (G₄S)₃ (15 amino acid residues, SEQ ID NO: 20), and a combination thereof.

Alternatively, the linker may be designed to have a thrombin recognition sequence (LVPRGS) or the like inserted thereinto so that Cap and IBD can be cleaved and dissociated at a desired timing. By designing in this way, for example, it is possible to, after the Cap-IBD fusion protein forms VLP, cleave and dissociate the IBD from the surface layer of the VLP.

Further, as described in the Examples of the present specification, GPGPGLVPRGSGPGPG (SEQ ID NO: 21), GPGPGGSGPGPG (SEQ ID NO: 22) obtained by removing the thrombin recognition sequence (LVPRGS) from the sequence of GPGPGLVPRGSGPGPG (SEQ ID NO: 21), and the like can also be used as the linker.

Note that in a case where the sequence of GPGPGGSGPGPG, which excludes the above thrombin recognition sequence, is used as a linker, it is possible to produce, for example, Cap-IBD (in this case, Cap-ZZ) consisting of the amino acid sequence represented by SEQ ID NO: 29 (or Cap-IBD that is encoded by a gene consisting of a polynucleotide consisting of a nucleotide sequence represented by SEQ ID NO: 30).

[Method for Producing Cap-IBD Fusion Protein]

A method for producing PCV2 VLP in accordance with an embodiment of the present invention includes the steps of: preparing an expression vector containing a nucleic acid which encodes the above-described Cap-IBD fusion protein; and introducing the expression vector into Escherichia coli to express the above-described Cap-IBD fusion protein.

A nucleic acid encoding Cap and a nucleic acid encoding the IBD (e.g., SpGB, PpLB, and Z) can be prepared, based on, for example, known nucleotide sequences (SEQ ID NOs: 2, 4, 6, 8, 10, and 12) by a conventional method including, for example, PCR cloning, chemical synthesis, and the like.

Further, a nucleic acid encoding the Cap-IBD fusion protein can be prepared by a conventional method including, for example, PCR subcloning, chemical synthesis, and the like.

An expression vector for producing the Cap-IBD fusion protein can be constructed by recombinant DNA technology using a known technique, and includes: the nucleic acid encoding the Cap-IBD fusion protein; and a regulatory sequence operably linked to the nucleic acid.

A transformed cell can be produced by introducing the above-described expression vector into Escherichia coli, which serves as a host cell. A method for transforming Escherichia coli is not particularly limited, and a known method such as calcium phosphate method and an electroporation method can be used.

By culturing the prepared transformed cell under a condition that allows expression of the Cap-IBD fusion protein, the Cap-IBD fusion protein is expressed as a soluble protein. The Cap-IBD fusion protein having been expressed as a soluble protein spontaneously forms VLP by spontaneous association (self-association) in the cell of the Escherichia coli expression host.

[Vaccine]

A vaccine in accordance with an embodiment of the present invention contains the above-described VLP as an active ingredient. Note that the content of the active ingredient in the vaccine is not particularly limited and can be set as appropriate.

In an embodiment of the present invention, the vaccine may contain, in addition to the VLP, a pharmaceutically acceptable additive(s) including, for example, a carrier, a lubricant, a preservative, a stabilizer, a wetting agent, an emulsifier, and the like.

The form of the vaccine is not particularly limited, and examples thereof include an injection, a liquid, a suspension, an emulsion, powder, a granule, a capsule, and the like. The administration form of the vaccine is not particularly limited, and may be peroral administration or may be parenteral administration (for example, subcutaneous administration, intramuscular administration, intracutaneous administration, nasal administration, intravenous administration, intraperitoneal administration, and the like).

A target animal to which the vaccine is administered is preferably a pig, although the target animal is not particularly limited, provided that it is a non-human animal which can be infected with PCV2.

An aspect of the present invention includes the following inventions:

<1> A fusion protein comprising a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain.

<2> The fusion protein described in <1> above, wherein the immunoglobulin-binding domain includes any one domain selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A.

<3> The fusion protein described in <1> or <2> above, wherein the immunoglobulin-binding domain is bound to a C-terminal of the capsid protein.

<4> The fusion protein described in any one of <1> to <3> above, wherein the immunoglobulin-binding domain has a structure in which two domains of any one type selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A are linked.

<5> The fusion protein described in any one of <1> to <4> above, wherein the capsid protein and the immunoglobulin-binding domain are bound via a linker.

<6> A virus-like particle formed by a fusion protein according to any one of <1> to <5> above.

<7> A vaccine comprising a virus-like particle described in <6> above.

<8> A method for producing a fusion protein comprising a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain, the method comprising the steps of: preparing an expression vector containing a nucleic acid which encodes the above-described fusion protein comprising the capsid protein of the porcine circovirus type 2 and the immunoglobulin binding domain; and introducing the above-described expression vector into Escherichia coli to express the above-described fusion protein.

<9> The method described in <8> above, wherein the immunoglobulin-binding domain includes any one domain selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A.

<10> The method described in <8> or <9> above, wherein the immunoglobulin-binding domain is bound to a C-terminal of the capsid protein.

<11> The method described in any one of <8> to <10> above, wherein the immunoglobulin-binding domain has a structure in which two domains of any one type selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A are linked.

<12> The method described in any one of <8> to <11> above, wherein the capsid protein and the immunoglobulin-binding domain are bound via a linker.

The present invention is not limited to the embodiments, but can be altered by a skilled person in the art within the scope of the claims. The present invention also encompasses, in its technical scope, any embodiment derived by combining technical means disclosed in differing embodiments.

EXAMPLES

The present invention will be more specifically described below with reference to Examples. Note, however, that the present invention is not limited to those Examples.

[1. Fusion of Cap and Solubilization-Promoting Tag (GST, SUMO)]

For all gene constructs other than Cap-GST, total gene synthesis was carried out using a nucleotide sequence (codon-optimized sequence) optimized for the Escherichia coli expression system. Note that a method for constructing a Cap-GST fusion gene is as follows.

By PCR subcloning, a nucleic acid encoding a fusion protein (Cap-GST) in which a solubility-promoting tag (GST) was fused to a C-terminal of Cap (SEQ ID NO: 41) via a peptide linker was prepared. Specifically, with use of Cap-ZZ as a template, a Cap-linker sequence was amplified by PCR using primeSTAR (registered trademark) (manufactured by Takara Bio Inc.) and a primer set (Cap F (SEQ ID NO: 37: 5′-tatacatatgacctatccgcgtcgccgcta-3′) and Cap link R (SEQ ID NO: 38: 5′-gtataggggaacccggacccggaccagagc-3′)). Further, with use of pET-41a (manufactured by Merck) as a template, a GST sequence was amplified by PCR using primeSTAR (registered trademark) and a primer set (GST F (SEQ ID NO: 39: 5′-gggtccgggttcccctatactaggttattg-3′) and GST R (SEQ ID NO: 40: 5′-agcgagctcttaatccgattttggaggatg-3′)). Subsequently, with use of the amplified Cap-linker sequence and GST sequence as templates, a Cap-linker-GST sequence was amplified by overlap PCR using primeSTAR (registered trademark) and a primer set (Cap F and GST R). With use of a ligation product of pJexpress441-01 (manufactured by ATUM) treated with restriction enzymes NdeI (manufactured by Takara Bio Inc.) and SacI (manufactured by Takara Bio Inc.) and the Cap-linker-GST sequence treated with the same restriction enzymes, NdeI and SacI, Escherichia coli DH5a strain (manufactured by Takara Bio Inc.) was transformed. From the transformed Escherichia coli DH5a strain, pJ4-CapGST was collected by an alkaline SDS method, and Escherichia coli BL21 strain (manufactured by Takara Bio Inc.) was transformed.

Expression of Cap-GST was attempted by culturing the obtained transformed cells with shaking at 37° C. in an LB medium containing ampicillin (manufactured by Nacalai Tesque, Inc.) until the turbidity (OD₆₀₀) become 0.4 to 0.6, then adding isopropyl-β-thiogalactopyranoside (IPTG) (manufactured by Sigma Aldrich) to a final concentration of 1 mM, and further carrying out culturing with shaking at 37° C. for 4 hours. As a result, Cap-GST did not express solubility at all and all became inclusion bodies.

Next, expression of a fusion protein (Cap-SUMO) in which a solubilization-promoting tag (SUMO) was fused to a C-terminal side of Cap was attempted in the same manner as above. A soluble fraction obtained by disrupting the bacterial cells which had been collected by centrifugation with BugBuster (registered trademark) (manufactured by Merck) contained Cap-SUMO at a level of about 3.2 mg/L culture.

In addition, a disrupted cell suspension obtained above was analyzed by gel filtration chromatography (Sephacryl S-300, manufactured by GE Healthcare). A fraction corresponding to the expected VLP position (at about 40 mL) were collected and photographed with an electron microscope. The observed VLPs were morphologically different from natural PCV2 particles, had a distorted particle shape, and had extremely non-uniform particle diameters (FIG. 7).

Note that GST is a generally widely-used solubilization-promoting tag having a molecular weight of 25.6 kDa, and have neither a β-grasp fold structure nor a three-helix bundle structure. In contrast, SUMO is a solubilization-promoting tag having a molecular weight of 11.0 kDa, which has been used often in recent years, and has a β-grasp fold structure.

From these results, GST had neither a Cap solubilization promoting function nor a VLP formation promoting function. Therefore, it was found that Cap cannot be expressed as a soluble protein in an Escherichia coli expression host simply by fusing Cap and a solubilization-promoting tag. Further, unlike GST, SUMO had the Cap solubilization promoting function, but did not have the VLP formation promoting function. The reason why GST and SUMO have different effects on the solubility of Cap in spite of the fact that both GST and SUMO are the solubilization-promoting tags is considered to be that, for example, molecular weights (GST: 25.6 kDa; SUMO: 11.0 kDa) and three-dimensional structures of GST and SUMO have effects.

[2. Fusion of Cap and IBDs (SpGB1-B2, PpLB1-B2)]

On the basis of the above-described result that SUMO having a β-grasp fold structure has a Cap solubilization promoting function, examination was performed by using a B domain of protein G (SpGB) and a B domain of protein L (PpLB) as other proteins having a β-grasp fold structure, like SUMO, to determine whether they had the Cap solubilization promoting function and the VLP formation promoting function.

A nucleic acid (SEQ ID NO: 24) encoding a fusion protein (Cap-SpGB1-B2: SEQ ID NO: 23) in which an IBD having a structure in which two SpGB were linked was fused to a C-terminal of Cap via a peptide linker was prepared, and expression of Cap-SpGB1-B2 was attempted in the Escherichia coli BL21 (DE3) strain by the same method as in Section 1. above. A soluble fraction obtained by disrupting the bacterial cells which had been collected by centrifugation with BugBuster (registered trademark) (manufactured by Merck) contained Cap-SpGB1-B2 at a level of about 1.5 mg/L culture.

In addition, a disrupted cell suspension obtained above was analyzed by gel filtration chromatography (Sephacryl S-300, manufactured by GE Healthcare). As a result of the analysis, a peak was observed at the expected VLP position (at about 40 mL), and the corresponding fraction was collected. As a result of photographing the collected fraction with an electron microscope, an image in which VLPs were uniformly dispersed in the aqueous solution was observed (FIG. 4). The observed VLPs strongly resembled in particle shape and in particle diameter to natural PCV2 particles, and had high uniformity.

The same operation was performed on a nucleic acid (SEQ ID NO: 26) encoding a fusion protein (Cap-PpLB1-B2: SEQ ID NO: 25) in which an IBD having a structure in which two PpLBs were linked was fused. A soluble fraction of a disrupted cell suspension contained Cap-PpLB1-B2 at a level of about 1.5 mg/L culture. In addition, as a result of gel filtration chromatography analysis of the disrupted cell suspension and photographing with an electron microscope, an image in which VLPs were uniformly dispersed in the aqueous solution was observed (FIG. 5). The observed VLPs strongly resembled in particle shape and in particle diameter to natural PCV2 particles, and had high uniformity.

From these results, it was revealed that SpGB and PpLB, which has a β-grasp fold structure similarly to SUMO, has, in addition to the Cap solubilization promoting function, an excellent VLP formation promoting function, unlike SUMO.

[3. Fusion of Cap and IBD (ZZ)]

In consideration of the result, obtained in Section 2. above, that the IBDs having a β-grasp fold structure have an excellent VLP formation promoting function, examination was performed to determine whether IBDs having a structure other than the β-grasp fold structure had the Cap solubilization promoting function and the VLP formation promoting function. Specifically, examination was performed by using a Z domain (Z) of protein A having a three-helix bundle structure as the IBD, to determine whether the Z domain (Z) of protein A had the Cap solubilization promoting function and VLP formation promoting function.

A nucleic acid (SEQ ID NO: 28) encoding a fusion protein (Cap-ZZ: SEQ ID NO: 27) in which an IBD having a structure in which two Zs were linked was fused to a C-terminal of Cap via a peptide linker was prepared, and expression of Cap-ZZ was attempted in the Escherichia coli BL21 (DE3) strain by the same method as in Section 1. above. In a soluble fraction obtained by disrupting the bacterial cells which had been collected by centrifugation with BugBuster (registered trademark) (manufactured by Merck), Cap-ZZ was expressed at a level (6.0 mg/L culture) exceeding the levels of Cap-SpGB1-B2 and Cap-PpLB1-B2.

In addition, a disrupted cell suspension obtained above was subjected to gel filtration chromatography analysis, and a fraction showing a peak corresponding to VLPs was collected. As a result of photographing the collected fraction with an electron microscope, an image in which VLPs were uniformly dispersed in the aqueous solution was observed (FIG. 6).

The observed VLPs strongly resembled in particle shape and in particle diameter to natural PCV2 particles, and had high uniformity.

From these results, it was revealed that Z, which has a three-helix bundle structure, has an extremely excellent Cap solubilization promoting function and an extremely excellent VLP formation promoting function.

Note that FIG. 3 schematically illustrates Cap and IBDs (SpGB1-B2, PpLB1-B2, ZZ) or the solubilization-promoting tag (SUMO) in the fusion proteins (Cap-SUMO, Cap-SpGB1-B2, Cap-PpLB1-B2, Cap-ZZ) collected above, and the arrangements of linkers (L) that exist between Cap and the IBDs or the solubilization-promoting tag (SUMO).

[4. Examination of the Effect of the Number of IBD Fusion Molecules on Cap Solubilization and VLP Formation]

In order to examine the effect of the number of IBD fusion molecules, a nucleic acid (SEQ ID NO: 32) encoding a fusion protein (Cap-Z: SEQ ID NO: 31) in which only one Z domain (Z) molecule of protein A was fused to Cap was prepared. As a result of expressing Cap-Z using the Escherichia coli BL21 (DE3) strain by the same method as in Section 1. above, the expression level was about 1.3 mg/L culture. This was found to be a decrease in solubility expression level in comparison with Cap-ZZ.

From this result, it is surmised that the stability of the fusion protein as a soluble protein was enhanced by linking two molecules of Z.

[5. Examination of the Effect of IBD Fusion Position on VLP Formation]

In order to examine the effect of an IBD fusion position on VLP formation, nucleic acids (SEQ ID NOs: 34 and 36, respectively) encoding fusion proteins (ZZ-Cap: SEQ ID NO: 33, Z-Cap: SEQ ID NO: 35) in which ZZ or Z was fused to an N-terminal of Cap via a peptide linker were prepared, and expression of ZZ-Cap and Z-Cap was attempted in the Escherichia coli BL21 (DE3) strain by the same method as in Section 1. above.

When ZZ-Cap and Z-Cap transformed cell disrupted solutions were electrophoresed (BN-PAGE (manufactured by Invitrogen)) in a non-denatured environment, VLP bands detected for Cap-ZZ and Cap-Z were remarkably light. This is presumed to be because ZZ-Cap and Z-Cap were subjected to a physical limitation that, in order for ZZ-Cap and Z-Cap to form VLP, ZZ and Z which are located on the N-terminal side need to be included within the lumen of the VLP.

[6. High-Temperature Treatment Stability Test: Examination of the Effect of High-Temperature Treatment on Maintenance of VLP]

The VLPs of the fusion proteins (Cap-SpGB1-B2, Cap-ZZ) obtained in Section 2. and Section 3. above were subjected to a high-temperature treatment stability test by the method below to examine the effect of a high-temperature treatment on the shape of the VLPs.

A VLP aqueous solution of Cap-ZZ was heat-treated at 52° C. for 30 minutes and at 97° C. for 30 minutes, and was then analyzed by gel filtration chromatography (Sephacryl S-300, manufactured by GE Healthcare).

FIG. 8 shows a chromatogram representing the analysis results obtained by gel filtration chromatography before and after heat treatment, for VLP of Cap-ZZ.

As shown in FIG. 8, for the VLP of Cap-ZZ, the chromatogram did not significantly change in shape even after 30 minutes of heat-treatment at both 52° C. and 97° C. The VLP of Cap-ZZ was confirmed to have high molecular stability against high-temperature treatment. Also for VLP of Cap-SpGB1-B2, a similar result was obtained.

Next, in order to evaluate the effect of the presence of IBDs on high-temperature treatment stability of VLP, Cap-IBDs (Cap-SpGB1-B2 and Cap-ZZ) were cleaved by thrombin treatment at a site of a thrombin recognition sequence (LVPR ↓ GS (↓: thrombin-specific cleavage site)) present in the peptide linker (L) to dissociate the IBDs from the VLP.

The VLP maintained its shape even after the IBDs exposed on the VLP surface layer were cleaved and dissociated.

For the VLP in which IBDs were cleaved and dissociated, a high-temperature treatment stability test was conducted in the same manner as above. FIG. 9 shows a chromatogram representing the analysis results obtained by gel filtration chromatography before and after heat treatment, for VLP of Cap-ZZ after subjected to dissociation of ZZ from the VLP surface layer of Cap-ZZ.

As shown in FIG. 9, in the chromatogram after heat treatment at 52° C. for 30 minutes, a peak corresponding to the VLP completely disappeared. Therefore, it is considered that the VLP after subjected to dissociation of ZZ was disintegrated by the heat treatment. Also for VLP of Cap-SpGB1-B2 after subjected to dissociation of SpGB1-B2 from the VLP surface layer of Cap-SpGB1-B2, a similar result was obtained.

From these results, it was confirmed that the above IBDs have a VLP disintegration suppressing function that imparts molecular stability against high-temperature treatment.

[7. Low-Temperature Treatment Stability Test: Examination of the Effect of Low-Temperature Treatment on Maintenance of VLP]

The VLPs of the fusion protein (Cap-ZZ) obtained in Section 3. above were subjected to a low-temperature treatment stability test by the method below to examine the effect of a low-temperature treatment on maintenance of the shape of the VLPs.

A PBS solution in which the VLPs of Cap-ZZ were dispersed was stored for 1 year under different temperature conditions of 4° C., −30° C., and −80° C. The PBS solution after storage was returned to room temperature and was observed with an electron microscope. Electron micrographs of the VLPs of Cap-ZZ after stored at each temperature for 1 year are shown in FIG. 10 (4° C.), FIG. 11 (−30° C.), and FIG. 12 (−80° C.).

As shown in FIGS. 10 to 12, the VLPs of Cap-ZZ maintain the shape and uniformity thereof even after low-temperature storage, and the phenomenon of reduction in the number of particles due to, for example, aggregation, shape change, and disintegration of the VLPs was not observed under any of the temperature conditions.

From these results, it was confirmed that the above IBD has a VLP disintegration suppressing function that imparts molecular stability against low-temperature treatment.

[8. Vaccine Antigen Preparation, as Well as Immunity and Attack Tests on Swine]

An aqueous solution containing the VLPs of the fusion protein (Cap-ZZ) obtained in Section 3. above and an oil adjuvant were mixed to prepare a vaccine antigen. 1 mL of this vaccine antigen contains approximately 20 μg of Cap antigen.

In addition, PBS and an oil adjuvant were mixed to prepare a Mock vaccine (pseudo-control).

To 4-week-old pigs purchased from a field farm, 1 mL of Cap-ZZ vaccine antigen or Mock vaccine was administered in the cervical muscle twice at an interval of two weeks (3 to 4 pigs per group). On the second week after the second administration, attack was performed by inoculating an attack emulsion containing 10⁵ FAID₅₀/mL of infectious PCV2 into the nasal cavity and the buttock muscle.

Blood was collected on the 7th, 10th, and 14th days after the attack, and after serum separation, DNA of PCV2 was extracted using the QIAamp (registered trademark) DNA Blood Mini Kit (manufactured by QIAGEN). In addition, on the 14th day after the attack, autopsy was performed, and tonsils, inguinal lymph nodes, and mesenteric lymph nodes were collected to prepare a 10% emulsion, and DNA was extracted by the same method as for serum.

Using SYBR Premix Taq (manufactured by Takara Bio Inc.) and a PCV2-specific primer set (CircoDF and CircoDR), the viral copy number of PCV2 (copies/mL or copies/g) was measured by real-time PCR method (Chromo4 system manufactured by Bio-Rad).

A plasmid with a known number of copies incorporating the Cap gene was used as a standard for preparing a calibration curve. The limit of quantitation of the viral copy number of PCV2 in this real-time PCR was 2,000 copies/mL in serum and 20,000 copies/g in tissues.

The result of measurement of the viral copy number in serum is shown in FIG. 13, and the result of measurement of the viral copy number in lymph nodes is shown in FIG. 14.

As shown in FIGS. 13 and 14, the Cap-ZZ immune group had a reduced viral copy number in serum (FIG. 13) and in each lymph node (FIG. 14) in comparison to the Mock group. This result indicates that the VLPs of the Cap-ZZ fusion protein can be used as an effective vaccine against PCV2 infection.

SUMMARY

Regarding the IBDs (SpGB, PpLB, and Z) used in the above Examples and the solubilization promoting tags (GST, SUMO), Table 1 below summarizes their known functions, three-dimensional structures, and results of tests for evaluation of various functions found by the present invention (Cap solubilization promoting function, VLP formation promoting function, VLP disintegration suppressing function (molecular stability imparting function), and vaccine function).

From the above results, it was found that fusing, with Cap of PCV2, IBDs, instead of GST or SUMO, which are well known as solubilization-promoting tags, makes it possible to mature such a protein as a vaccine antigen. More interestingly, it was confirmed that these IBDs have the VLP disintegration suppressing function, that is, the function of imparting molecular stability against physicochemical load.

INDUSTRIAL APPLICABILITY

The present invention is useful in the field of pharmaceuticals, particularly in the field of vaccines. 

1. A fusion protein comprising a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain.
 2. The fusion protein according to claim 1, wherein the immunoglobulin-binding domain includes any one domain selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A.
 3. The fusion protein according to claim 1, wherein the immunoglobulin-binding domain is bound to a C-terminal of the capsid protein.
 4. The fusion protein according to claim 1, wherein the immunoglobulin-binding domain has a structure in which two domains of any one type selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A are linked.
 5. The fusion protein according to claim 1, wherein the capsid protein and the immunoglobulin-binding domain are bound via a linker.
 6. A virus-like particle formed by a fusion protein according to claim
 1. 7. A vaccine comprising a virus-like particle according to claim
 6. 8. A method for producing a fusion protein comprising a capsid protein of porcine circovirus type 2 and an immunoglobulin-binding domain, the method comprising the steps of: preparing an expression vector containing a nucleic acid which encodes the fusion protein comprising the capsid protein of the porcine circovirus type 2 and the immunoglobulin binding domain; and introducing the expression vector into Escherichia coli to express the fusion protein.
 9. The method according to claim 8, wherein the immunoglobulin-binding domain includes any one domain selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A.
 10. The method according to claim 8, wherein the immunoglobulin-binding domain is bound to a C-terminal of the capsid protein.
 11. The method according to claim 8, wherein the immunoglobulin-binding domain has a structure in which two domains of any one type selected from the group consisting of a B domain of protein G, a B domain of protein L, and a Z domain of protein A are linked.
 12. The method according to claim 8, wherein the capsid protein and the immunoglobulin-binding domain are bound via a linker. 